Recombinant pox virus for immunization against MUC1 tumor-associated antigen

ABSTRACT

Recombinant pox viruses capable of expressing an immunogenic fragment of the MUC1 tumor-associated antigen are disclosed. The recombinant viruses can be used as vaccines to prevent the establishment of or treat tumors or pre-tumorous cells expressing the MUC1 tumor-associated antigen. The vaccines can be provided as an admixture comprising: (1) a recombinant pox virus encoding the immunogenic fragment of the MUC1 tumor-associated antigen, and (2) a recombinant pox virus encoding a T-cell co-stimulatory factor. The vaccine admixture can be used, e.g., to prevent establishment of tumors or pre-tumorous cells expressing the MUC1 tumor-associated antigen. The MUC1 specific cytotoxic T-cells can be isolated and expanded and used in a method for treating a host having a tumor expressing MCU1 positive tumor cells.

The following application is a continuation of U.S. Ser. No. 09/366,670filed on Aug. 3, 1999, now abandoned which is a continuation ofPCT/US98/03693 filed on Feb. 24, 1998, now abandoned, which claimedbenefit under 35 U.S.C. 119 of U.S. Provisional Application 60/038,253filed on Feb. 24, 1997.

BACKGROUND OF THE INVENTION

The immunotherapeutic approach to the treatment of cancer is based onthe observation that human tumor cells express a variety oftumor-associated antigens (TAAs) that are not typically expressed innormal tissues. These antigens, which include viral tumor antigens,cellular oncogene proteins, and tumor-associated differentiationantigens, can serve as targets for the host immune system and elicitresponses which result in tumor destruction. This immune response ismediated primarily by lymphocytes; T cells in general and class IMHC-restricted cytotoxic T lymphocytes in particular play a central rolein tumor rejection. Hellstrom, K. E., et al., (1969) Adv. Cancer Res.12:167–223; Greenberg, P. D. (1991) in Advances in Immunology, vol. 49(Dixon, D. J., ed.), pp. 281–355, Academic Press, Inc., Orlando, Fla.Unfortunately, as evidenced by the high incidence of cancer in thepopulation, the immune response to neoplastic cells often fails toeliminate tumors. The goal of active cancer immunotherapy is theaugmentation of anti-tumor responses, particularly T cell responses, inorder to effect complete tumor destruction.

Most attempts at active immunization against cancer antigens haveinvolved whole tumor cells or tumor fragments. However, the cloning ofTAAs recognized by CD8+ T cells has opened new possibilities for theimmunotherapy of cancer based on the use of recombinant or syntheticanti-cancer vaccines. Boon, T., et al., (1994) Annu. Rev. Immunol.12:337–365; Brithcard, V., et al., (1993) J. Exp. Med. 178:489–495; Cox,A. L., et al., (1994) Science 264:716–719; Houghton, A. N. (1994) J.Exp. Med. 180:1–4; Pardoll, D. M. (1994) Nature 369:357–358; Kawakami,Y., et al., (1994) Proc. Natl. Acad. Sci. U.S.A. 91:3515–3519; Kawakami,Y., et al., (1994) Proc. Natl. Acad. Sci. U.S.A. 91:6458–6462.

DF3/MUC1 (MUC1) is a cell surface glycoprotein that is overexpressed inbreast, ovarian, and pancreatic tumors. The major extracellular portionof MUC1 is composed of tandem repeat units of 20 amino acids whichcomprise immunogenic epitopes. The full length major extracellular MUC1protein is composed of up to 100 tandem repeat units of 20 amino acidscontaining 0-glycosylation sites which act as a framework for theformation of a highly glycosylated structure, which is highlyimmunogenic.

The term “tandem repeat unit” of MUC1 refers to the 20 amino acidrepeated sequence of MUC1 (see, e.g., Gendler, S. J., et al (1990) J.Biol. Chem. 265:15286–15293).

(SEQ ID NO:1) GSTAPPAHGVTSAPDTRPAP

There is an abnormal glycosylation pattern found in carcinoma cellsmaking the tumor-derived mucin antigenically distinct from normal mucin.Monoclonal antibodies specific for these peptide epitopes as well astheir unique sugar side chains can identify >90% of breast tumors.

See Kufe, D., et al. (1984) Hybridoma 223–32; Taylor-Papadimitriou, J.,et al. (1994) Trends Biotechnol. 12:227–33; Fontenot, J. D., et al.(1993) Cancer Res. 53:5386–94; Siddiqui J., et al. (1988) Proc. Natl.Acad. Sci. USA 85:2320–3; Merlo et al. (1989) Cancer Res. 49:6966–6971;and Abe, M., et al. (1989) Biochem Biophys Res Commun 165:644–9.

Accordingly, using the MUC1 tumor-associated antigen (TAA) has beenproposed in developing cancer vaccines, particularly against tumorsexpressing MUC1. Multiple copies of tandem repeats are required foroptimal native conformation and immunogenicity (see Fontenot et al.,supra). A comparison of synthetic peptides containing 3, 4, or 5.25tandem repeats of MUC1 revealed that the 5.25-copy version most closelymimicked the native structure of MUC1 and showed the most anti-mucinreactivity (Kotera et al. (1994) Cancer Res. 54:2856–2860). Previousrecombinant vaccinia viruses containing the MUC1 gene with numeroustandem repeats were found to be unstable; homologous recombinationresulted in deletion of most of the repeats, reducing the efficacy ofthe vaccine. See, e.g., Acres, R. B., et al. (1993) J. Immunother.14:136–43; Bu, D., et al. (1993) J. Immunother. 14:127–35; Hareuveni,M., et al. (1990) Proc. Natl. Acad. Sci. USA. 87:9498–502; and Finn O.J. et al. infra.

The use of recombinant vaccinia viruses for anti-tumor immunotherapy hasbeen discussed. (Hu, S. L., Hellstrom, I., and Hellstrom K. E. (1992) inVaccines: New Approaches to Immunological Problems (R. W. Ellis, ed) pp.327–343, Butterworth-Heinemann, Boston.) Anti-tumor responses have beenelicited using recombinant pox viruses expressing TAAs such ascarcinoembryonic antigen (CEA) and prostrate specific antigen (PSA).(Muraro, R., et al., (1985) Cancer Res. 4S:5769–5780); (Kantor, 3., etal. (1992) J. Natl. Cancer Inst. 84:1084–1091); (Robbins, P. F., et al.(1991) Cancer Res. 51:3657–3662) (Kantor, 3., et al. (1992) Cancer Res.52:6917–6925.) No toxicity with these vectors was observed.

In general, viral vaccines are believed to mediate tumor rejection byactivating class I MHC-restricted T-cells, particularly cytotoxic Tlymphocytes (CTLs). T-cell activation is often potentiated by providinga suitable immunomodulator, for example a T-cell co-stimulatory factorsuch as those of the B7 gene family. See e.g., Greenberg, P. D. (1991)in Advances in Immunology, Vol. 49 (Dixon, D. J., ed.), pp. 281–355,Academic Press, Inc., Orlando, Fla.; Fox B. A. et al. (1990) J. Biol.Response Mod. 9:499–511.

It would be useful to have a recombinant pox virus encoding a MUC1fragment containing a number of tandemly repeated sequences that willgenerate a cytotoxic T-cell response to MUC1, but which is stable,undergoing minimal excision as a result of homologous recombination inthe gene encoding MUC1. It would also be useful to provide therecombinant pox virus in a vaccine format which is capable ofpotentiating T-cell activity against such tumors, particularlyestablished or pre-existing tumors expressing the MUC1 TAA.

SUMMARY OF THE INVENTION

The present invention relates to recombinant pox viruses encoding a MUC1fragment, vaccines, and methods of using the recombinant pox viruses andvaccines to generate an immune reaction to MUC1 which can be used toprevent or treat tumors expressing MUC1 TAAs.

The recombinant pox virus of the present invention contains a geneencoding an immunogenic MUC1 fragment of 5 to 25 tandem repeats of the20 amino acid unit, preferably 7–15 tandem repeats, more preferablyabout 7–10 tandem repeats, still more preferably about 10 tandemrepeats, which when expressed, can vaccinate a mammal against tumors orpre-tumorous cells expressing the MUC1 TAA. This MUC1 gene fragment isstable, maintaining the tandem repeat copy number at around 10 copies.

In some preferred embodiments the DNA segment encoding the tandemrepeats is altered from the native pattern by using alternative codonsto reduce homology between the repeats. For example, amino acidstypically have two or more codons that will encode the same residue(e.g., glycine is encoded by GGT, GGA, GGG, or GGC). By usingalternative codons encoding the same amino acid one can further reducethe possibility of undesired recombination events. Additionally, one canalso introduce some conservative amino acid changes into differentgroups of the tandem repeats to further reduce undesired recombination(e.g., glycine/serine, valine/leucine), taking care not to alter apeptide epitope that would reduce its immunogenicity.

The immunogenic “mini-MUC1 fragments” do not undergo significant geneticdeletion, thereby improving stability. Moreover, the fragment impartssufficient immunogenic specificity for MUC1 immunogenicity. The effectcan further be enhanced by providing a T-cell co-stimulatory factor suchas B7 and/or a cytokine such as interleukin-2 (IL-2), particularly forthe treatment of established or pre-existing tumors expressing the MUC1TAA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Western blot showing expression of a MUC1 polypeptide,referred to as mini-MUC1 or miMUC1, from rV-MUC1 infected MC38 cells.

FIGS. 2A and 2B are graphs showing expression of MUC1 proteins in MC38cells. FIG. 2B: MC38 cells transduced with a retroviral vectorcontaining the mini-MUC1 gene and stained with the DF3 anti-MUC1antibody. FIG. 2A: Non-transduced MC38 cells.

FIG. 3 is a graph illustrating primary CTL activity followingimmunization with an admixture of rV-MUC1 and rV-B7.

FIG. 4 is a graph showing prevention of MUC1-positive pulmonarymetastases by immunization with rV-MUC1.

FIG. 5 is a graph illustrating treatment of established MUC1-positivepulmonary metastases by immunization with an admixture of rV-MUC1 andrV-B7.

FIG. 6 is a graph illustrating survival of mice immunized with anadmixture of rV-MUC1 and rV-B7.

DETAILED DESCRIPTION OF THE INVENTION

Recombinant pox viruses of the present invention encode a mini-MUC1fragment that can induce an immunogenic response to MUC1, preferably acytotoxic T cell response, and can thus serve as effective vectors forvaccination against tumors or pre-tumorous cells expressing the MUC1TAA. Vaccine efficacy can substantially be enhanced by providing animmune modulator such as a T-cell co-stimulatory factor such as B7-1,B7-2 and/or a cytokine such as IL-2. This is preferred in treatingestablished or pre-existing tumors expressing the MUC1 TAA.

A recombinant pox virus of the present invention can be derived from anaturally-occurring or designated wild-type pox virus strain. The poxvirus will be a DNA cytoplasmic pox virus which does not integrate intoa host cell genome. Exemplary of such pox viruses are suipox (e.g.,swine pox), capripox, leporipox, avipox (e.g., fowl pox, canary pox) andorthopox (e.g., vaccinia, ectromelia, rabbit pox). Representative poxviruses can be obtained from the ATTC such as fowlpox (VR-229) andswinepox (VR-363). A particularly preferred pox virus is vacciniaavailable from the ATCC as the Wyeth Strain (VR-325).

In one preferred embodiment, the recombinant pox viruses of the presentinvention made from such pox viruses are characterized as beingsubstantially avirulent. That is, it does not cause disease in thedesired target cell or tissue. The selected pox virus may have a hostrange that does not include the target host species, therebysubstantially restricting propagation of the virus in the host. Forexample, swinepox can be used as a pox virus vector in accordance withthe present invention when the host is a primate such as a human.Alternatively, a modified strain of the pox virus can be used to conferavirulence in the normal host range of the pox virus.

Exemplary pox viruses for use in accordance with the present inventionare suitable orthopox such as vaccinia viruses, avipox such as fowl pox,canary pox and pigeon pox and suipox such as swine pox. Several suitablestrains of vaccinia virus are available, e.g., in an attenuated formsuch as the MVA or Wyeth strain. These vaccinia strains aresubstantially attenuated in their normal host range (see e.g., Smith, K.A., et al. (1993) Vaccine 11:43–53).

A preferred example of a vaccinia virus suitable for making arecombinant vaccinia virus in accordance with the present invention isthe Wyeth strain such as the vTBC33 derivative of the Wyeth strainprovided in Example 1. A preferred avipox is fowlpox.

An immunogenic MUC1 fragment can be inserted into a suitable pox virusby conventional recombinant techniques to produce the presentrecombinant pox viruses. For example, as will be explained in moredetail in Reference Example 1 and the examples which follow, a DNA donorplasmid vector including a DNA insert encoding an immunogenic MUC1fragment can be constructed to provide recombination between DNAsequences flanking the insert in the donor plasmid vector and homologoussequences present in the virus. Accordingly, a recombinant virusencoding the immunogenic MUC1 fragment is formed therefrom. Othertechniques can be used to make the recombinant virus encoding theimmunogenic MUC1 fragment including use of a unique restrictionendonuclease site that is naturally present or artificially inserted inthe parental viral vector (see e.g., Mackett, et al., Proc. Natl. Acad.Sci. USA 79:7415–7419 (1982); and U.S. Pat. No. 5,093,258).

More particularly, the immunogenic MUC1 fragment can be inserted byconventional methods into the DNA donor vector such as those suitablefor use in a prokaryote such as E. coli. The donor vector will furtherinclude viral DNA which is homologous to a segment of pox virus DNA atthe site to which insertion of the MUC1 fragment is desired. DNAencoding the immunogenic MUC1 fragment can be inserted into the DNAdonor vector adjacent to suitable control elements in the vector such aspromoter, enhancer, ribosome binding, and leader sequences. The DNA soinserted into the donor vector is typically positioned to provideflanking viral DNA (e.g., vaccinia HindIIIM fragments) on both ends ofthe insert. As stated previously, the flanking viral DNA will generallybe homologous to a DNA sequence flanking a region of the pox virus DNAto which insertion is desired. Preferably, the homologous flanking viralDNA sequence will be 100% homologous to the region of the pox virus DNAto which insertion is desired. Exemplary DNA donor vectors generallyinclude an origin of replication such as the E. coli origin ofreplication, and a marker such as an antibiotic resistance gene forselection and propagation in a suitable host such as E. coli. Theresulting DNA donor vector is then propagated by growth within asuitable prokaryotic host cell, isolated and purified if desired.

The DNA donor vector including the immunogenic MUC1 fragment to beinserted into a desired pox virus is generally transfected into asuitable cell culture, e.g., a primate cell line or chick embryofibroblasts, that is infected with the pox virus. Recombination betweenhomologous DNA in the DNA donor vector and the pox virus genome forms arecombinant pox virus modified by the presence of the immunogenic MUC1fragment. Preferably, the site of pox virus insertion does notsubstantially affect the viability of the recombinant pox virus. Viralviability can be readily tested by, e.g., viral plaque assay or a DNAreplication assay involving tagging newly synthesized DNA with adetectably-labeled nucleotide (e.g. ³H-thymidine). Typically, viralviability will be assessed by comparing the viability of the recombinantpox virus to that of a control pox virus (i.e., no inserted DNA).

As noted above, the immunogenic MUC1 fragment is inserted into asuitable region (insertion region) of a pox virus so that virusviability is not substantially affected. The skilled artisan can readilyidentify such regions in the pox virus by, for example, randomly testingsegments of virus DNA for regions that allow recombinant formationwithout affecting virus viability of the recombinant. One region thatcan readily be used and is present in many viruses is the thymidinekinase (TK) gene. For example, it has been found in all pox virusgenomes examined (e.g., leporipoxvirus: Upton, et al., J. Virology,60:920 (1986) (shope fibroma virus); capripoxvirus: Gershon, et al., J.Gen. Virol., 70:525 (1989) (Kenya sheep-1); orthopoxvirus: Weir, et al.,J. Virol., 46:530 (1983) (vaccinia); Esposito, et al., Virology,135:561(1984) (monkeypox and variola virus); Hruby, et al., PNAS,80:3411(1983) (vaccinia); Kilpatrick, et al., Virology, 143:399 (1985)(Yaba monkey tumor virus); avipoxvirus: Binns, et al., J. Gen. Virol.69:1275(1988) (fowlpox); Boyle, et al., Virology, 156:355(1987)(fowlpox); Schnitzlein, et al., J. Virological Methods, 20:341(1988)(fowlpox, quailpox); entomopox (Lytvyn, et al., J. Gen. Virol.73:3235–3240 (1992)].

In vaccinia, in addition to the TK region, other insertion regionsinclude, for example, HindIII M.

In fowlpox, in addition to the TK region, other insertion regionsinclude, for example, BamHI J [Jenkins, et al., AIDS Research and HumanRetroviruses 7:991–998 (1991)] the EcoRI-HindIII fragment, BamHIfragment, EcoRV-HindIII fragment, BamHI fragment and the HindIIIfragment set forth in EPO Application No. 0 308 220 A1. [Calvert, etal., J. of Virol. 67:3069–3076 (1993); Taylor, et al., Vaccine 6:497–503(1988); Spehner, et al., (1990) and Boursnell, et al., J. Gen. Virol.71:621–628 (1990)].

In swinepox preferred insertion sites include the thyrnidine kinase generegion and the HindIIIC region.

In addition to the requirement that the gene be inserted into aninsertion region, successful expression of the inserted gene by themodified poxvirus requires the presence of a promoter operably linked tothe desired gene, i.e., in the proper relationship to the inserted gene.The promoter must be placed so that it is located upstream from the geneto be expressed. Promoters are well known in the art and can readily beselected depending on the host and the cell type you wish to target. Forexample in poxviruses, pox viral promoters should be used, such as thevaccinia 7.5K, or 40K or fowlpox Cl. Artificial pox promoter constructscontaining appropriate promoter sequences can also be used. Enhancerelements can also be used in combination to increase the level ofexpression. Furthermore, the use of inducible promoters, which are alsowell known in the art, are preferred in some embodiments.

For example, it is possible to make a DNA vector construct in which thepromoter is modulated by an external factor or cue, and in turn tocontrol the level of polypeptide being produced by the vectors byactivating that external factor or cue. For example, heat shock proteinsare proteins encoded by genes in which the promoter is regulated bytemperature. The promoter of the gene which encodes the metal-containingprotein metallothionine is responsive to Cd+ ions. Incorporation of thispromoter or another promoter influenced by external cues also makes itpossible to regulate the production of the proteins.

The pox vectors of the present invention contain a DNA fragment encodinga MUC1 fragment, sometimes referred to as mini-MUC. The MUC1 genefragment will encode a sufficient portion of MUC1 to generate an immunereaction to MUC1, but does not undergo extensive excision as a result ofhomologous recombination. Preferably, the fragment is approximately 5 to25 MUC1 tandem repeat units, more preferably between approximately 7 to15 MUC1 tandem repeat units, and most preferably about 7 to 12 MUC1tandem repeat units. An especially preferred immunogenic MUC1 fragmentis about 10 MUC1 tandem repeat units. Preferred fragments have the humanMUC1 DNA sequence. A preferred MUC1 DNA sequence is the human MUC1 cDNAsequence having the repeat units disclosed, e.g., by Gendler et al.supra. While the sequence reported by Merlo et al., supra, is 10 MUC1tandem repeat units, a sample based on this was only about 7 tandemrepeat units. This sample is more fully described in the examples.

In some preferred embodiments the DNA segment encoding the tandemrepeats is altered from the native pattern in such a manner as to reduceduplications of the codons. For example, amino acids typically have twoor more codons that will encode the same residue (e.g., glycine isencoded by GGT, GGA, GGG, or GGC). By using other codons encoding thesame amino acid one can further reduce the possibility of undesiredrecombination events. Additionally, one can also introduce someconservative amino acid changes into different groups of the tandemrepeats to further reduce undesired recombination (e.g., glycine/serine,valine/leucine), taking care not to alter a peptide epitope that wouldreduce its immunogenicity.

Preferably, the 60 bp tandem repeat sequence can be altered to minimizenucleotide homology without changing the amino acid sequence. Forexample the first tandem repeat in miMUC1 can be left unaltered asfollows:

       1   2   3   4   5   6   7   8   9   10       GGC TCC ACC GCC CCCCCA GCC CAC GGT GTC        G   S   T   A   P   P   A   H   G   V      11  12  13  14  15  16  17  18  19  20       ACC TCG GCC CCG GACACC AGG CCG GCC CCG        T   S   A   P   D   T   R   P   A   P *(SEQID NO:2)

The second, third, and fourth tandem repeats can then be altered in thethird base of threonine codons 3, 11 and 16 using ACG, ACT, and ACA,respectively. These repeats can also be altered in alanine codons 4, 7,13, and 19, using GCG, GCA, and GCT respectively. Similar third-basealterations can be incorporated at numerous codons in each of the tandemrepeats to minimize homologous recombination among the repeats. Oneexample of MUC1 repeat sequences using wobbled codons to minimizehomology while retaining repeated amino acid sequence is set forth belowin Table A.

TABLE A R1 GGC TCC ACC GCC CCC CCA GCC CAC GGT GTC ACC TCG GCC CCG GACACC AGG CCG GCC CCG (SEQ ID NO:2) R2 GGC AGT ACT GCA CCA CCG GCA CAT GGCGTA ACA TCA GCA CCT GAT ACA AGA CCT GCA CCT (SEQ ID NO:4) R3 GGA TCC ACCGCG CCG CCT GCG CAC GGA GTG ACG TCG GCG CCC GAC ACG CGC CCC GCT CCC (SEQID NO:5) R4 GGG TCA ACA GCT CCT CCC GCT CAT GGG GTT ACT TCT GCT CCA GATACT CGC CCA GCT CCA (SEQ ID NO:6) R5 GGT TCG ACG GCC CCC CCT GCT CAC GGTGTA ACA TCC GCC CCG GAT ACC AGA CCG GCC CCT (SEQ ID NO:7) R6 GGC AGC ACCGCA CCG CCC GCA CAC GGG GTC ACA AGC GCG CCA GAC ACT CGA CCT GCG CCA (SEQID NO:8) R7 GGA AGT ACC GCT CCA CCT GCA CAC GGG GTC ACA AGC GCG CCA GACACT CGA CCT GCG CCA (SEQ ID NO:9) R8 GGG TCG ACT GCC CCT CCG GCG CAT GGTGTG ACC TCA GCT CCT GAC ACA AGG CCA GCC CCA (SEQ ID NO:10) R9 GGT TCAACG GCA CCT CCA GCA CAC GGA GTC ACG TCT GCA CCC GAC ACC CGT CCA GCT CCG(SEQ ID NO:11) R10 GGT AGT ACA GCG CCA CCC GCA CAT GGC GTC ACG AGC GCTCCG GAT ACG AGA CCG GCG CCT (SEQ ID NO:12) G   S   T   A   P   P   A   H   G   V   T   S   A   P   D   T   R   P   A   P(SED ID NO:1)

One can use the various sequences in any combination. Further, one doesnot need to use all 10 repeats.

Nucleotide homology can also be reduced by introducing changes to theamino acid sequence, preferably conservative amino acid substitutionsinto some of the tandem repeats. Immunogenic epitopes such as (SEQ IDNO:3) PDTRPAP would preferably be left intact, but valine codon 10 couldbe changed to leucine codons CTT, CTC, CTA, and CTG in differentrepeats.

An immunogenic MUC1 fragment according to the invention can be made by avariety of conventional methods. For example, the fragment can be madeby cloning a desired portion of the full-length human MUC1 DNA sequence(see e.g., Merlo, et al., supra; and Abe, M., et al., supra).Restriction enzymes can be used to cleave the desired fragment. Theimmunogenic MUC1 DNA fragment can also be prepared by amplification bythe Polymerase Chain Reaction (i.e., PCR). Use of cloning and PCRamplification techniques to make an immunogenic MUC1 fragment isdisclosed in Example 1 which follows.

An immunogenic mini-MUC1 fragment in accordance with the presentinvention can be inserted into a suitable pox virus to produce arecombinant pox virus which encodes the intact fragment and isreasonably stable. Expression of the immunogenic MUC1 fragment can bereadily determined by several methods, including assaying samples of asuitable target cell or tissue by SDS-PAGE gel electrophoresis followedby Coomassie blue or silver staining; Western blot using DF3 antibody,or other suitable immunological technique such as ELISA.

Live recombinant viruses expressing an immunogenic cell encoded tumorassociated antigen can be used to induce an immune response againsttumor cells which express the protein. These recombinant viruses may beadministered by scarification, as was conventionally done for small poxvaccination, or by other routes appropriate to the recombinant virusused. These may include among others, intramuscular, intradermal,subcutaneous, and intravenous routes. Vaccination of a host organismwith live recombinant vaccinia virus is followed by replication of thevirus within the host.

For parenteral administration, the recombinant vectors will typically beinjected in a sterile aqueous or non-aqueous solution, suspension oremulsion in association with a pharmaceutically-acceptable carrier suchas physiological saline. Kits containing the vector and the means forinjection can be used. The kit preferably contains instructionsdescribing how to use the vector. In one embodiment, the kit contains avector modified to include an immunomodulator or a separate vectorcontaining the immunomodulator as described below. In addition the kitcan contain an adjuvant.

A specific immune response to a tumor associated antigen can begenerated by administering between about 10⁵–10⁹ pfu of the recombinantpox virus, constructed as discussed above to a host; more preferably oneuses ≧10⁷ pfu. The preferred host is a human. At least one intervalthereafter, which is preferably one to three months later, the immuneresponse is boosted by administering additional antigen to the host.More preferably there is at least a second “boost” preferably at leastone to three months after the first boost, more preferably 6–12 monthsafter the first boost. The boosting antigen may be administered usingthe same pox virus vector, or as a whole protein, an immunogenic peptidefraction of the protein, another recombinant viral vector, or DNAencoding the protein or peptide. Preferably, different pox viral vectorsare used. For example, vaccinia may be followed by an avipox such asfowlpox, or vice versa. Cytokines, e.g., IL-2, IL-6, IL-12, IL-15, orco-stimulatory molecules, e.g., B7.1, B7.2, may be used as biologicadjuvants. The cytokines can be administered systemically to the host.Either cytokines or co-stimulatory molecules can be co-administered viaco-insertion of the genes encoding the molecules into the recombinantpox vector or a second recombinant poxvirus which is admixed with therecombinant poxvirus expressing the TAA.

Adjuvants include, for example, RIBI Detox (Ribi Immunochemical), QS21(Aquila), incomplete Freund's adjuvant or many others.

Alternatively, it will sometimes be useful to use a recombinant poxvirus encoding the immunogenic MUC1 fragment which has been modified toinclude an immunomodulator, for example, DNA encoding a T-cellco-stimulatory factor and/or a cytokine such as interleukin (IL) (e.g.,IL-2, IL-4, IL-10, IL-12), an interferon (IFN) (e.g., IFN-γ),granulocyte macrophage colony stimulating factor (GM-CSF) or anaccessory molecule (e.g. ICAM-1). The construction of such multivalentvectors such as pox viral vectors is within the level of skill in theart based upon the present disclosure. In some cases, co-expression ofthe immunomodulatory agent such as the T-cell co-stimulatory factor andthe immunogenic fragment of MUC1 by multiple vectors may be desirable.It may be desirable to administer a substantially pure preparation of,e.g., the immunomodulator to boost vaccine efficacy.

In preferred embodiments after initial administrations of the viralvector by one pox a different pox virus, preferably from a different poxfamily will be used for the following administrations (i.e. boosts). Forexample, initial administrations by vaccinia or avipox would preferablybe followed by boosts from an avipox or vaccina, respectively, or by asuipox.

Although initially generally less preferred in most cases, it may bedesirable to use another DNA or RNA virus or vector to insert animmunogenic MUC1 DNA fragment into a subject host. Such an approach maybe useful where multiple boosts are used and the subject is at risk ofdeveloping an antigenic reaction to the host pox vector. Exemplary ofsuch vectors are DNA or RNA viruses such as retroviruses, adenoviruses,herpes viruses or DNA-based vectors (see generally, Cepko et al., Cell37:1053–1062 (1984); Morin et al., Proc. Natl. Acad. Sci. USA84:4626–4630 (1987); Lowe et al., Proc. Natl. Acad. Sci. USA,84:3896–3900 (1987); Panicali & Paoletti, Proc. Natl. Acad. Sci. USA,79:4927–4931(1982); Mackett et al., Proc. Natl. Acad. Sci. USA,79:7415–7419 (1982)). In an alternative embodiment, one would primefirst with a non-pox viral vector expressing mini-MUC1, or a DNA segmentencoding mini-MUC1, followed by boosting, wherein at least one boostinvolves the use of pox vectors.

Further contemplated uses of the recombinant pox viruses disclosedherein include use in the production of antibodies, particularlymonoclonal antibodies that are capable of specifically binding theimmunogenic MUC1 fragments. More specifically, it can be desirable toproduce the antibodies, e.g., to detect mucin glycosylation in tumor andpre-tumorous cells in vitro and in vivo. The antibodies may be preparedby a variety of standard methods well-known to those skilled in the art.For example, cells expressing an immunogenic MUC1 fragment can beadministered to an animal to induce production of polyclonal antibodies.Alternatively, monoclonal antibodies which specifically bind animmunogenic MUC1 fragment can be prepared using hybridoma technology(see, e.g., Kohler et al., Nature 256: 495 (1975); Hammerling et al., InMonoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y. (1981)).

Cytotoxic T-cells specific for an immunogenic MUC1 fragment can beestablished from peripheral blood mononuclear cells (PBMC) obtained froma host immunized as discussed above. For example, PBMC can be separatedby using Lymphocyte Separation Medium gradient (Organon Teknika, Durham,N.C., USA) as previously described Boyum, et al., Scand J. Clin LabInvest 21: 77–80 (1968). Washed PBMC are resuspended in a completemedium, for example, RPMI 1640 (GIBCO) supplemented with 10% pool humanAB serum (Pel-Freeze Clinical System, Brown Dear, Wis., USA), 2 mMglutamine, 100 U/ml penicillin and 100 μg/ml of streptomycin (GIBCO).PBMC at a concentration of about 2×10⁵ cells in complete medium in avolume of, for example, 100 μl are added into each well of a 96-wellflat-bottom assay plate (Costar, Cambridge, Mass., USA). The immunogenicMUC1 fragment can be added to the cultures in a final concentration ofabout 50 μg/ml and incubated at 37° C. in a humidified atmospherecontaining 5% CO₂ for 5 days. After removal of the media containing thefragment, the cultures are provided with fresh human IL-2 (10 U/ml)after 5 days and replenished with IL-2 containing medium every 3 days.Primary cultures are restimulated with the immunogenic MUC1 fragment (50μg/ml) on day 16. 5×10⁵ irradiated (4,000 rad) autologous PBMC are addedin a volume of about 50 μl complete medium as antigen-presenting cells(APC). About five days later, the cultures are provided with human IL-2containing medium as described previously. Cells are restimulated for 5days at intervals of 16 days.

The cytotoxic T-cells can be cultured in accordance with known methodsand then injected back into the host by a variety of means. Generally,between 1×10⁵ and 2×10¹¹ cytotoxic T-cells per infusion are administeredin, for example, one to three infusions of 200 to 250 ml each over aperiod of 30 to 60 minutes. After the completion of the infusions, thepatient may be treated with recombinant interleukin-2 with a dose of720,000 IU per kilogram of body weight intravenously every eight hours;some doses can be omitted depending on the patient's tolerance for thedrug. In addition, after infusion, additional recombinant pox virus orimmunogenic MUC1 fragment containing T-cell eliciting epitope(s) may beadministered to the patient to further expand the T-cell number. Theantigen or epitope may be formulated with an adjuvant and/or may be in aliposomal formulation.

The cytotoxic T-cells can also be modified by introduction of a viralvector containing a DNA encoding TNF and reintroduced into a host in aneffort to enhance the anti-tumor activity of the cells.

REFERENCE EXAMPLE 1

Pox Viruses

A number of pox viruses have been developed as live viral vectors forthe expression of heterologous proteins. Representative vaccinia virusstrains such as Wyeth and MVA have been disclosed previously. (Cepko etal., Cell 37:1053–1062 (1984); Morin et al., Proc. Natl. Acad. Sci. USA84:4626–4630 (1987); Lowe et al., Proc. Natl. Acad. Sci. USA,84:3896–3900 (1987); Panicali & Paoletti, Proc. Natl. Acad. Sci. USA,79:4927–4931(1982); Mackett et al., Proc. Natl. Acad. Sci. USA,79:7415–7419 (1982)). Representative fowlpox and swinepox virus areavailable through the ATCC under accession numbers VR-229 and VR-363,respectively. The Wyeth strain of vaccinia is available through the ATCCunder accession number VR-325.

DNA Vectors for In Vivo Recombination with a Parent Virus

Genes that code for desired carcinoma associated antigens are insertedinto the genome of a pox virus in such a manner as to allow them to beexpressed by that virus along with the expression of the normalcomplement of parent virus proteins. This can be accomplished by firstconstructing a DNA donor vector for in vivo recombination with a poxvirus.

In general, the DNA donor vector contains the following elements:

(i) a prokaryotic origin of replication, so that the vector may beamplified in a prokaryotic host;

(ii) a gene encoding a marker which allows selection of prokaryotic hostcells that contain the vector (e.g., a gene encoding antibioticresistance);

(iii) at least one gene encoding a desired protein located adjacent to atranscriptional promoter capable of directing the expression of thegene; and

(iv) DNA sequences homologous to the region of the parent virus genomewhere the foreign gene(s) will be inserted, flanking the construct ofelement (iii).

Methods for constructing donor plasmids for the introduction of multipleforeign genes into pox virus are described in WO91/19803, the techniquesof which are incorporated herein by reference. In general, all DNAfragments for construction of the donor vector, including fragmentscontaining transcriptional promoters and fragments containing sequenceshomologous to the region of the parent virus genome into which foreigngenes are to be inserted, can be obtained from genomic DNA or cloned DNAfragments. The donor plasmids can be mono-, di-, or multivalent (i.e.,can contain one or more inserted foreign gene sequences).

The donor vector preferably contains an additional gene which encodes amarker which will allow identification of recombinant viruses containinginserted foreign DNA. Several types of marker genes can be used topermit the identification and isolation of recombinant viruses. Theseinclude genes that encode antibiotic or chemical resistance (e.g., seeSpyropoulos et al., J. Virol., 62:1046 (1988); Falkner and Moss., J.Virol., 62:1849 (1988); Franke et al., Mol. Cell. Biol., 5:1918 (1985),as well as genes such as the E. coli lacZ gene, that permitidentification of recombinant viral plaques by colorimetric assay(Panicali et al., Gene, 47:193–199 (1986)).

Integration of Foreign DNA Sequences into the Viral Genome and Isolationof Recombinants

Homologous recombination between donor plasmid DNA and viral DNA in aninfected cell results in the formation of recombinant viruses thatincorporate the desired elements. Appropriate host cells for in vivorecombination are generally eukaryotic cells that can be infected by thevirus and transfected by the plasmid vector. Examples of such cellssuitable for use with a pox virus are chick embryo dermal (CED) cells,HuTK143 (human) cells, and CV-1 and BSC-40 (both monkey kidney) cells.Infection of cells with pox virus and transfection of these cells withplasmid vectors is accomplished by techniques standard in the art(Panicali and Paoletti, U.S. Pat. No. 4,603,112, WO89/03429).Alternatively, the donor DNA can be directly ligated into the parentalvirus genome at a unique restriction site (Scheiflinger, et al. (1992)Proc. Natl. Acad. Sci. (USA) 89:9977–9981).

Following in vivo recombination or ligation, recombinant viral progenycan be identified by one of several techniques. For example, if the DNAdonor vector is designed to insert foreign genes into the parent virusthymidine kinase (TK) gene, viruses containing integrated DNA will beTK⁻ and can be selected on this basis (Mackett et al., Proc. Natl. Acad.Sci. USA, 79:7415 (1982)). Alternatively, co-integration of a geneencoding a marker or indicator gene with the foreign gene(s) ofinterest, as described above, can be used to identify recombinantprogeny. One preferred indicator gene is the E. coli lacZ gene:recombinant viruses expressing β-galactosidase can be selected using achromogenic substrate for the enzyme (Panicali et al., Gene, 47:193(1986)).

Characterizing the Viral Antigens Expressed by Recombinant Viruses

Once a recombinant virus has been identified, a variety of methods canbe used to assay the expression of the polypeptide encoded by theinserted gene. These methods include black plaque assay (an in situenzyme immunoassay performed on viral plaques), Western blot analysis,radioimmunoprecipitation (RIPA), enzyme immunoassay (EIA), or functionalassay such as CTL assay.

EXAMPLE 1

Construction of Recombinant Vaccinia Virus Encoding MUC1 Gene Sequences

A. Mini-MUC1 Gene Vector

The human DF3/MUC1 cDNA was constructed from two cloned cDNA segments[Merlo, et al., supra; Abe, M. et al., surpa]. A 1.8 kb EcoRI fragmentof MUC1 cDNA reported as containing 10 tandem repeats and its 3′ uniquesequence was inserted into Bluescript™ plasmid (Stratagene, La Jolla,Calif.) at the EcoRI site and designated pBs-MUC1. The 5′ end of theMUC1 gene was generated from another MUC1 clone by PCR using MUC1specific primers. The 200 base pair amplification fragment was insertedinto pBs-MUC1 at the HindIII and HindIII sites creating pBS-miMUC1containing the “mini” MUC1 gene (sometimes referred to herein as“miMUC1”). However, DNA sequence analysis of the miMUC1 gene confirmedthat this gene contained the appropriate signal and start site, but not10 tandem repeats. Instead it contained 7 repeats that showed somevariation. The DNA sequence of the repeated portion of the miMUC1 geneis set forth below in Table B.

The deduced amino acid sequence of the repeat region predicted fromnucleotide sequence analysis of MUC1 gene is set forth below in Table C.

Moreover, the 3′ coding sequence actually differs from that reported byMerlo, supra, but conforms to the 3′ sequence reported by Gendler,supra. The entire coding sequence of the miniMUC1 gene is shown in TableD.

TABLE B R1                           GGC TCC ACC GCC CCC CCA GCC CAC GGTGTC ACC TCG CCG GCC GAC (SEQ ID NO:2)                           ACC AGGCCG GCC CCG R2                           GGC TCC ACC GCC CCC CCA GCC CACGGT GTC ACC TCG GCC CCG GAC (SEQ ID NO:2)                           ACCAGG CCG GCC CCG R3                           GGC TCC ACC GCC CCC CCA GCCCAC GGT GTC ACC TCG GCC CCG GAC (SEQ ID NO:2)                          ACC AGG CCG GCC CCG R4                          GGC TCC ACC GCC CCC CCA GCC CAC GGT GTC ACCTCG CCG GCC GAC (SEQ ID NO:2)                           ACC AGG CCG GCCCCG R5 GGC TCC ACC GCA CCC CCA GCC CAC GGT GTC ACC TCG GCC CCG GAC ACCAGG CGG GCC CCG GGC (SEQ ID NO:13)                           TCC ACC CCGGCC CCG R6                           GGC TCC ACC GCC CCC CCA GCC CAT GGTGTC ACC TCG GCC CCG GAC (SEQ ID NO:14)                           ACC AGGCCC GCC TTG R7                           GGC TCC ACC GCC CCT CCA GTC CACAAT GTC ACC TCG GCC (SEQ ID NO:15)

TABLE C Repeat 1             G S T A P P A H G V T S A P D T R P A P(SEQ ID NO:1) Repeat 2             G S T A P P A H G V T S A P D T R P AP (SEQ ID NO:1) Repeat 3             G S T A P P A H G V T S A P D T R PA P (SEQ ID NO:1) Repeat 4             G S T A P P A H G V T S A P D T RP A P (SEQ ID NO:16) Repeat 5: G S T P A P G S T A P P A H G V T S A P DT R P A P (SEQ ID NO:1) Repeat 6:             G S T A P P A H G V T S AP D T R P A P (SEQ ID NO:17) Repeat 7:             G S T A P P V H N V TS A (SEQ ID NO:18)

TABLE D (SEQ ID NO:19)ATGACACCGGGCACCCAGTCTCCTTTCTTCCTGCTGCTGCTCCTCACAGTGCTT  M  T  P  G  T  Q  S  P  F  F  L  L  L  L  L  T  V  LACAGCTACCACAGCCCCTAAACCCGCAACAGTTGTTACGGGTTCTGGTCATGCA  T  A  T  T  A  P  K  P  A  T  V  V  T  G  S  G  H  AAGCTCTACCCCAGGTGGAGAAAAGGAGACTTCGGCTACCCAGAGAAGTTCAGTG  S  S  T  P  G  G  E  K  E  T  S  A  T  Q  R  S  S  VCCCAGCTCTACTGAGAAGAATGCTGTGAGTATGACAAGCTTGATATCGAATTCC  P  S  S  T  E  K  N  A  V  S  M  T  S  L  I  S  N  SGGTGTCCGGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGAC  G  V  R  G  S  T  A  P  P  A  H  G  V  T  S  A  P  DACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCC  T  R  P  A  P  G  S  T  A  P  P  A  H  G  V  T  S  ACCGGACACCAGGCCGGCCCCGGGCTCCACCGCCCCCCCAGCCCACGGTGTCACC  P  D  T  R  P  A  P  G  S  T  A  P  P  A  H  G  V  TTCGGCCCCGGACACCAGGCCGGCCCCGGGCTCCACCGCACCCCCAGCCCACGGT  S  A  P  D  T  R  P  A  P  G  S  T  A  P  P  A  H  GGTCACCTCGGCCCCGGACACCAGGCGGGCCCCGGGCTCCACCCCGGCCCCGGGC  V  T  S  A  P  D  T  R  R  A  P  G  S  T  P  A  P  GTCCACCGCCCCCCCAGCCCACGGTGTCACCTCGGCCCCGGACACCAGGCCGGCC  S  T  A  P  P  A  H  G  V  T  S  A  P  D  T  R  P  ACCGGGCTCCACCGCCCCCCCAGCCCATGGTGTCACCTCGGCCCCGGACAACAGG  P  G  S  T  A  P  P  A  H  G  V  T  S  A  P  D  N  RCCCGCCTTGGGCTCCACCGCCCCTCCAGTCCACAATGTCACCTCGGCCTCAGGC  P  A  L  G  S  T  A  P  P  V  H  N  V  T  S  A  S  GTCTGCATCAGGCTCAGCTTCTACTCTGGTGCACAACGGCACCTCTGCCAGGGCT  S  A  S  G  S  A  S  T  L  V  H  N  G  T  S  A  R  AACCACAACCCCAGCCAGCAAGAGCACTCCATTCTCAATTCCCAGCCACCACTCT  T  T  T  P  A  S  K  S  T  P  F  S  I  P  S  H  H  SGATACTCCTACCACCCTTGCCAGCCATAGCACCAAGACTGATGCCAGTAGCACT  D  T  P  T  T  L  A  S  H  S  T  K  T  D  A  S  S  TCACCATAGCACGGTACCTCCTCTCACCTCCTCCAATCACAGCACTTCTCCCCAG  H  H  S  T  V  P  P  L  T  S  S  N  H  S  T  S  P  QTTGTCTACTGGGGTCTCTTTCTTTTTCCTGTCTTTTCACATTTCAAACCTCCAG  L  S  T  G  V  S  F  F  F  L  S  F  H  I  S  N  L  QTTTCCTTCCTCTCTCGAAGATCCCAGCACCGACTACTACCAAGAGCTGCAGAGA  F  P  S  S  L  E  D  P  S  T  D  Y  Y  Q  E  L  Q  RGACATTTCTCAAATGTTTTTGCAGATTTATAAACAAGGGGGTTTTCTGGGCCTC  D  I  S  Q  M  F  L  Q  I  Y  K  Q  G  G  F  L  G  LTCCAATATTAAGTTCAGGCCAGGATCTGTGCTGGTACAATTGACTCTGGCCTTC  S  N  I  K  F  R  P  G  S  V  L  V  Q  L  T  L  A  FCGAGAAGGTACCATCAATGTCCACGACGTGGAGACACAGTTCAATCAGTATAAA  R  E  G  T  I  N  V  H  D  V  E  T  Q  F  N  Q  Y  KACGGAAGCAGCCTCTCGATATAACCTGACGATCCCAGACGTCAGCGTGAGTGAT  T  E  A  A  S  R  Y  N  L  T  I  P  D  V  S  V  S  DGTGCCATTTCCTTTCTCTGCCCAGTCTGGGGCTGGGGTGCCAGGCTGGGGCATC  V  P  F  P  F  S  A  Q  S  G  A  G  V  P  G  W  G  IGCGCTGCTCCTGCTGGTCTGTGTTCTGTTGCGCTGGCCATTGTCTATCTCATT  A  L  L  L  L  V  C  V  L  V  A  L  A  I  V  Y  L  IGCCTTGGCTGTCTGTCAGTGCCGCCGAAAGAACTACGGGCAGCTGGACATCTTT  A  L  A  V  C  Q  C  R  R  K  N  Y  G  Q  L  D  I  FCCAGCCCGGGATACCTACCATCCTATGAGCGAGTACCCCACCTACCACACCCAT  P  A  R  D  T  Y  H  P  M  S  E  Y  P  T  Y  H  T  HGGGCGCTATGTCCCCCCTAGCAGTACCGATCGTAGCCCCTATGAGAAGGTTTCT  G  R  Y  V  P  P  S  S  T  D  R  S  P  Y  E  K  V  SGCAGGTAATGGTGGCAGCAGCCTCTCTTACACAAACCCAGCAGTGGCAGCCACT  A  G  N  G  G  S  S  L  S  Y  T  N  P  A  V  A  A  T TCTGCCAACTTGTAG  S  A  N  LB. Recombinant Vaccinia Virus

The miMUC1 gene described above was inserted 3′ to the vaccinia 40Kearly/late promoter and flanked by sequences from the Hind III M regionof the vaccinia genome. The resulting plasmid, designated pT2041,contained the miMUC1 gene under the control of the vaccinia virus 40Kearly/late promoter flanked by DNA sequences from the Hind III M regionof the vaccinia genome. These flanking sequences included the vacciniaK1L host range gene required for growth of vaccinia virus on rabbitkidney RK13 cells (ATCC CCL37). A plaque-purified derivative of theWyeth strain of vaccinia was used as the parental virus (designatedvTBC33), lacked a functional K1L gene and thus could not efficientlyreplicate on RK13 cells. See e.g., Gritz, L., et al. (1990) J. Virol.64:5948–57; Gillard, S., et al. (1986) Proc. Natl. Acad. Sci. USA.83.5573–7; and Smith, K A., et al., supra.

Generation of recombinant vaccinia virus was accomplished via homologousrecombination between vaccinia sequences in the vTBC33 genome and thecorresponding sequences in pT2041 in vaccinia-infected RK13 cellstransfected with pT2041. Recombinant virus, designated vT46(rV-MUC1),was selected by growth on RK13 cells (ATCC CCL37). Virus stocks wereprepared by clarifying infected RK13 cell lysates followed bycentrifugation through a 36% sucrose cushion.

The selection and screening of rV-MUC1 was done by growth in RK13 cells.The recombinant vaccinia rV-MUC1 was isolated as a single recombinantclone and purified by two rounds of plaque purification. The miMUC1 geneinsertion into the vaccinia virus genome HindIII site by homologousrecombination was confirmed by Southern analysis with ³²P radiolabeledmiMUC1 gene as a probe. The Southern analysis indicated that thevaccinia virus had not deleted any portions of the gene, in contrast tothe deletions reported with full-length MUC1 genes (see Bu, D., et al.supra).

A plasmid similar to pT2041 was constructed that contained the lacZ genein addition to the mini-MUC1 gene; this plasmid was designated pT2068.The plasmid DNA pT2068 was deposited with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, under theconditions of the “Budapest Treaty on the International Recognition ofthe Report of Microorganisms for the Purpose of Patent Procedure.” Thedeposit was given ATCC Designation 97893. If the culture dies or isdestroyed during the effective term, it will be replaced if a patentissues. If a patent issues, the strain will be maintained for 30 yearsfrom the date of deposit, or five years after the most recent request,whichever is longer.

PT2068 was used to construct a recombinant vaccinia virus containingmini-MUC1 using a colorimetric screen for β-gal.

A recombinant vaccinia virus strain expressing the human co-stimulatorymolecule B7-1 (designated rV-B7) has been disclosed. The virus was grownin spinner cultures of HeLa cells, directly pelleted by centrifugation,and purified over 20%–40% sucrose gradients (Hodge, J. W., et al. (1994)Cancer Res. 54:5552–5; Earl, P. L., et al. (1993) Generation ofrecombinant vaccinia viruses, Vol. 2, Suppl. 21, 16.17.1–16.18.10. NewYork: John Wiley & Sons).

EXAMPLE 2

Characterization of Recombinant Vaccinia Virus With miMUC1 Insert

A. Southern Blot Hybridization Analysis

BSC-1 cells (ATCC CC 126) were infected at an MOI of 10 with eithermiMUC1 recombinant vaccinia virus (designated rV-MUC1) or V-Wyeth. Theviral DNA extraction, restriction endonuclease digestion and Southernblotting was performed as previously described (see e.g., Kaufman, H.,et al. (1991) Int. J. Cancer. 48:900–7). The results indicated that themiMUC1 gene was stably inserted into the HindIII region of the vacciniagenome.

B. Western Analysis of Protein Expression and Stability

Parallel confluent BSC-1 cells were infected with either parental wildtype vaccinia virus (V-Wyeth), or rV-MUC1 at an MOI of 5 in Dulbecco'smodified Eagle's medium containing 2.5% FBS. After an overnightinfection, cells in one plate were scraped and lysed in hypotonic lysisbuffer (100 mM Tris-HCI pH 8.0, 100 mM NaCI, 0.5% NP-40, and 0.2 mMPMSF). The infected cells in the corresponding parallel plate werescraped and high titer virus preparations were derived as previouslydescribed (Earl, P. L., et al. (1993) Generation of recombinant vacciniaviruses, Vol. 2, Supplement 21, 16.17.1–16.18.10. New York: John Wiley &Sons). The resulting virus was used to infect parallel confluent BSC-1cells as before. This cycle was repeated to generate protein extractsfrom cells infected with rV-MUC1 that had gone through 2, 3, 4, and 5passages of viral replication. Cell lysates were electrophoresed on anSDS-10% acrylamide gel. Proteins were electroblotted ontonitrocellulose, blocked, incubated with DF3 antibody (Kufe, D., et al.supra) for 4 hours at room temperature, washed and incubated with goatanti-mouse phosphatase labeled secondary antibody (Kirkegaard and Perry,Gaithersburg, Md.) and developed according to the manufacturer'sinstructions.

Stable expression of MUC1 after 2, 3, 4 or 5 passages of viralreplication of rV-MUC1 was confirmed by Western analysis (FIG. 1).Incubation of protein extracted from rV-MUC1 infected cells from passage2 (FIG. 1, lane 2) with the monoclonal antibody DF3 revealed a broad150–175 kD band. Similarly, incubation of protein extracted from cellsinfected with viral passages 3, 4, and 5 with DF3 (lanes 3, 4, and 5)revealed identical bands ranging from 150–175 kD. Lane 1 containspurified MUC1 protein of approximately 3OOkD. This finding is consistentwith reports indicating the apparent molecular mass of theseglycoproteins, which appear heterogeneous as a result of 0-linkedglycosylation in the tandem repeats (Sekine, H., et al. (1985) J.Immunol. 135:3610–5). Uninfected or V-Wyeth infected cells were negativefor the expression of MUC1 by Western blot using DF3 MAb.

EXAMPLE 3

Construction and Characterization of Recombinant Vaccinia VirusContaining MUC1 and B7.1

The miMUC1 gene and the human B7.1 gene were each ligated to vacciniapromoters. The promoter-gene cassettes were then inserted into a plasmidvector containing the E. coli lacZ gene flanked by DNA sequences fromthe HindIII M region of the vaccinia genome. The resulting plasmid,designated pT2043, contains the B7.1 gene under the control of thevaccinia virus 30K promoter (located at the HindIII M insertion site;Perkus et al. (1985) Science 229: 981–984), the MUC1 gene under thecontrol of the vaccinia virus 40K early/late promoter (Gritz et al.,supra), and the lacZ gene under the control of the fowlpox Cl promoter(Jenkins et al., (1991) AIDS Res. Human Retrovirus 7:991–998), allflanked by DNA sequences from the HindIII M region of the vacciniagenome. A plaque-purified derivative of the Wyeth strain of vaccinia wasused as the parental virus in the construction of recombinant vacciniavirus. The generation of recombinant vaccinia virus was accomplished viahomologous recombination between vaccinia sequences in the Wyethvaccinia genome and the corresponding sequences in pT2043 invaccinia-infected RK₁₃ cells transfected with pT2043. Recombinant virus,designated vT2043, was identified using a chromogenic substrate forβ-galactosidase (Bluo-Gal™). Viral plaques expressing lacZ appeared blueagainst a clear background. Positive plaques were picked from the cellmonolayer and their progeny were further propagated. Repeated rounds ofplaque isolation and replating in the presence of Bluo-Gal resulted inthe purification of the desired recombinant. Virus stocks were preparedby clarifying infected RK₁₃ cell lysates followed by centrifugationthrough a 36% sucrose cushion. Insertion of the MUC1 and B7.1 genes intothe vaccinia genome was confirmed by Southern analysis using MUC1 andB7.1 gene probes. Expression of MUC1 and B7.1 protein was demonstratedby Western analysis using antibodies specific for each protein. Morepreferably, another cell line such as the monkey kidney cell line CV-1(ATCC CCL 70) or chick embryo dermal (CED) cells would be used forvaccine production.

EXAMPLE 4

Construction and Characterization of Recombinant Avipox Virus ContainingMUC1 and B7.1

The miMUC1 gene is inserted into a plasmid vector containing thevaccinia 40K promoter and the E. coli lacZ gene flanked by DNA sequencesfrom the BamHI J region of the fowlpox genome. The resulting plasmidcontains the miMUC1 gene under the control of the vaccinia virus 40Kearly/late promoter (Gritz et al., supra), and the lacZ gene under thecontrol of the fowlpox Cl promoter (Jenkins et al., supra), all flankedby DNA sequences from the BamHI J region of the fowlpox genome. Theparental virus used for the generation of this recombinant virus is theUSDA licensed live fowlpox vaccine POXVAC-TC (Schering-PloughCorporation). The generation of recombinant vaccinia virus isaccomplished via homologous recombination between fowlpox sequences inthe POXVAC-TC fowlpox genome and the corresponding sequences in theplasmid vector in fowlpox-infected chick embryo dermal cells (CED),prepared as described (Jenkins et al., supra), transfected with theplasmid vector. Recombinant virus is identified using a chromogenicsubstrate for β-galactosidase (Bluo-Gal™). Viral plaques expressing lacZappear blue against a clear background. Positive plaques are picked fromthe cell monolayer and their progeny are further propagated. Repeatedrounds of plaque isolation and replating in the presence of Bluo-Galresult in the purification of the desired recombinant. Virus stocks areprepared by clarifying infected CED cell lysates followed bycentrifugation through a 20% sucrose cushion. Insertion of the MUC1 geneinto the fowlpox genome is confirmed by Southern analysis using a MUC1gene probe. Expression of MUC1 protein is demonstrated by Westernanalysis using antibodies specific for MUC1.

Construction and characterization of a recombinant fowlpox viruscontaining both MUC1 and B7.1 is accomplished by inserting apromoter-B7.1 cassette into the plasmid described above, and by carryingout the manipulations described above.

Recombinant canary pox viruses containing MUC1 or MUC1 and B7.1 areconstructed and characterized in an analogous fashion using canary poxas the parental virus (Taylor et al. (1991) Vaccine 9:190–193; Paoletti,U.S. Pat. No. 5,505,941).

EXAMPLE 5

1. Transfection and Transduction of the miMUC1 Gene in pLNSX

A 2 kb XhoI/XbaI restriction endonuclease fragment from pBs-miMUC1 wasisolated and the ends repaired with DNA polymerase 1-Klenow fragment andligated into the Stu I site of the retroviral vector pLNSX. ThepLNSX-mIMUC1 gene was transfected into the PA317 packaging cell line byLipofectin (GIBCO/BRL) according to the manufacturer's instructions.Cells were harvested, plated onto 60 mm dishes, and incubated with200–500 μg/ml G418 for three weeks. Clones of PA317 cells containing themiMUC1 gene were identified by Northern blot analysis of total RNAisolated from G418 resistant clones using the XbaI/XhoI DNA fragment ofthe miMUC1 gene as a radioactive probe. The retroviral supernatants ofMUC1-transduced PA317 cells were collected and used to transduce MC38cells in the presence of polybrene (8 μg/ml). Following transduction,MC38 cells were selected by cloning G418 resistant colonies andselection by FACs analysis using DF3 antibody. The resultant MUC1positive cell line was designated MC38/MUC1. Those cells were shown tobe negative for B7-1 expression by flow cytometry.

The amphotrophic packaging cell line PA317 was obtained from Dr. RobertBassin (National Cancer Institute, NIH, Bethesda, Md.).

The MC38 murine colonic adenocarcinoma cell line (20) was obtained fromthe laboratory of Dr. Steve Rosenberg (National Cancer Institute, NIH,Bethesda, Md.).

EXAMPLE 6

FACS Analysis of Recombinant Protein Expression

Cell surface expression of MUC1 on MC38/MUC1 cells was analyzed byimmunofluorescence. Cells were harvested and incubated at 4° C. for 30minutes with 1 μg/ml DF3 MAb in 5% FBS-DPBS, followed by incubation withfluorescein-labeled goat anti-mouse IgG (Kirkegaard and Perry) for 30minutes at 4° C. Analysis was performed with a FACScan (Becton-DickinsonMountain View, Calif.).

Surface expression of MUC1 glycoprotein in MUC1-transduced MC38 cellswas examined by flow cytometry. FIGS. 2A and 2B illustrate thatuntransduced MC38 cells (FIG. 2A) do not react with DF3 MAb (98.5% ofthe cells are negative with a mean fluorescence of 20). However, MC38cells transduced with the MUC1 gene (FIG. 2B) react strongly with theDF3 antibody (87.5% of the cells are positive with a mean fluorescenceof 400). These studies thus demonstrate that MC38 tumor cells transducedwith the miMUC1 gene (MC38/MUC1) express the MUC1 molecule.

EXAMPLE 7

Anti-Tumor Activity of Recombinant Vaccinia Virus Vaccine

A. Cytotoxicity Assay. To analyze the effect of rV-MUC1 or rV-MUC1/rV-B7vaccination on MUC1 specific cytotoxic activity, splenic lymphocytesfrom mice inoculated with rV-MUC1 or the mixture of rV-MUC1 and rV-B7were tested for their ability to lyse murine adenocarcinoma cells thatwere negative (MC38) or positive for MUC1 (MC38/MUC1) (Kantor, 3., etal. supra). Briefly, spleens were removed and mechanically dispersedthrough 70 mm cell strainers (Falcon, Becton Dickinson, Franklin Lakes,N3) to isolate single cell suspensions. Erythrocytes and dead cells wereremoved by centrifugation over a Ficoll-Hypaque gradient (density=1.119g/ml) (Sigma Chemical Co., St. Louis, Mo.). MC38 cells and MC38/MUC1cells were prepared for use as targets in a standard indium releaseassay as described previously (Hodge, J. W., et al. (1995) Cancer Res.55:3598–603). Tumor cells (2×10⁶ cells) were radiolabeled with 50 μCi of¹¹¹In oxyquinoline solution (Amersham, Arlington Heights, Ill.) for 30minutes at 37° C. and dispensed (10⁴ cells/50 μl) into each well of96-well U-bottom plates (Costar, Cambridge, Mass.). T-cells were addedto effector to target (E:T) ratios of 100:1–12.5:1 in 96 well U-bottomedplates (Costar) and incubated for 16 hours at 37° C. with 5% CO₂. Afterincubation, supernants were collected using a Supernatant CollectionSystem (Skatron, Sterling, Va.) and radioactivity was quantitated usinga gamma counter. (Cobra Autogamma, Packard, Downers Grove, Ill.). Thepercentage of specific release of ¹¹¹In was determined by the standardequation: % specificlysis=[(experimental−spontaneous)/(maximum−spontaneous)]×100.

FIG. 3 shows the results of an experiment in which groups of mice wereinoculated with an admixture of 10⁷ PFU rV-MUC1 and 10⁷ PFU V-Wyeth(squares), or an admixture of 10⁷ PFU rV-MUC1 and 10⁷ PFU rV-B7(circles). All groups were inoculated with an admixture of 10⁷ PFUrV-MUC1 and 10⁷ PFU V-Wyeth after 14 and 28 days. Seven days followingthe final immunization, cytolytic activity was quantified against MC38cells (MUC1 negative; closed symbols) or MC38/MUC1 cells (MUC1 positive;open symbols). T-cells from mice inoculated three times withrV-MUC1/V-Wyeth or one time with rV-MUC1/rV-B7 followed by twoinoculations with rV-MUC1/V-Wyeth did not lyse the MUC1 negative MC38targets (closed symbols), but did lyse the MUC1 positive MC38/MUC1targets (open symbols). This MUC1 specific lysis was E:T ratiodependent.

B. Prevention of MUC1 Positive Pulmonary Metastases

Groups of C57BL/6 mice were inoculated subcutaneously with either (a) anadmixture of 10⁷ PFU rV-B7 and 10⁷ PFU V-Wyeth; (b) an admixture of 10⁷PFU rV-MUC1 and 10⁷ PFU V-Wyeth; or (c) an admixture of 10⁷ PFU rV-MUC1and 10⁷ PFU rV-B7. After two weeks, mice in the first group wereinoculated with 2×10⁷ PFU V-Wyeth; while the remaining two groups wereinoculated with an admixture of 10⁷ PFU rV-MUC1 and 10⁷ PFU V-Wyeth. Twoweeks later, mice were challenged intravenously with 2×10⁶ MC38/MUC1tumor cells. Mice were euthanized 28 days following tumor transplant andexperimental pulmonary metastatic nodules as defined by Wexler (Wexler,H., et al. (1966) J. Natl. Cancer Inst. 36:641–645) were stained. Thesemetastatic nodules were enumerated in a blind fashion, and lungs withnodules too numerous to count were assigned an arbitrary value of >250.

FIG. 4 illustrates the efficacy of rV-MUC1 in this experimental tumormodel. Mice inoculated with rV-B7/V-Wyeth were all positive for lungmetastases ( 8/10 mice had greater than 250 nodules). In contrast, 90%of mice inoculated with rV-MUC1/V-Wyeth and boosted with rV-MUC1/V-Wyethremained free of pulmonary metastases ( 1/10 mice had 6 nodules).Similarly, 90% of mice receiving the same immunization scheme with theaddition of rV-B7 in the first immunization remained free of pulmonarymetastases.

C. Therapy of Established MUC1 Positive Pulmonary Metastases

C57BL/6 mice were challenged intravenously with 2×10⁶ MC38/MUC1 tumorcells. After 3 days, mice were randomized and inoculated subcutaneouslywith either (a) 2×10⁷ PFU V-Wyeth; (b) an admixture of 10⁷ PFU rV-B7 and10⁷ PFU V-Wyeth; (c) an admixture of 10⁷ PFU rV-MUC1 and 10⁷ PFUV-Wyeth; or (d) an admixture of 10⁷ PFU rV-MUC1 and 10⁷ PFU rV-B7. Sevendays later, mice in the first two groups were inoculated intravenouslywith 2×10⁷ PFU V-Wyeth, while the remaining two groups were inoculatedintravenously with an admixture of 10⁷ PFU rV-MUC1 and 10⁷ PFU V-Wyeth.Seven days later, following this inoculation, mice were inoculated athird time in a similar fashion. Mice were euthanized 28 days followingtumor transplant and pulmonary metastatic nodules were stained andenumerated as above. Identically treated groups were followed forsurvival. Kaplan-Meier plots and Mantel-Cos (Logrank) tests were used tocompare survival of mice belonging to different treatment groups.

FIG. 5 shows the efficacy of rV-MUC1 in a therapeutic setting. Miceinoculated with V-Wyeth or rV-B7/V-Wyeth were all positive for lungmetastases ( 9/10 and 7/10, respectively had greater than 250 nodules.)Although all mice inoculated 3 times with rV-MUC1/V-Wyeth were positivefor lung nodules, the number of metastases was comparatively low ( 7/10with <50 nodules). In contrast, 30% of mice inoculated withrV-MUC1/rV-B7 and boosted with rV-MUC1/V-Wyeth remained free ofpulmonary metastases, while the remaining mice all had less than 20 lungnodules.

FIG. 6 depicts a different parameter of therapy of established MUC1positive pulmonary metastases in which a parallel group of mice wasinoculated identically to those above and monitored for survival. In theexperiment shown in FIG. 6, groups of 10 mice were transplantedintravenously with 2×10⁶ MC38/MUC1 tumor cells, and tumors were allowedto establish for 3 days. Mice were inoculated every 7 days as in FIG. 5.Immunization sequences were: V-Wyeth: V-Wyeth: V-Wyeth (open triangles);rV-B7/V-Wyeth: V-Wyeth: V-Wyeth (closed circles); rV-MUC1/V-Wyeth:rV-MUC1/V-Wyeth: rV-MUC1/V-Wyeth: rV-MUC1/V-Wyeth: rV-MUC1/V-Wyeth(closed squares); and rV-MUC1/rV-B7: rV-MUC 1/V-Wyeth: rV-MUC1/V-Wyeth(open circles). Vaccination of mice with V-Wyeth or rV-B7/V-Wyeth andboosting with V-Wyeth had no effect on mouse survival, with 100%mortality by 50–56 days post tumor challenge. In contrast, inoculationof mice three times with rV-MUC1/V-Wyeth resulted in a significantimprovement of survival time (p=0.0009−<0.0001). Furthermore,immunization of mice with rV-MUC1/rV-B7 followed by two boosts withrV-MUC1/V-Wyeth resulted in 100% survival of mice (p<0.0001). It thusappears that the administration of rV-MUC1 can significantly improve thesurvival of mice bearing MUC1 positive tumors, but only the admixture ofrV-MUC1 with rV-B7 can completely eradicate MUC1 expressing tumors.Lungs of these animals were examined at day 65 and were free of tumornodules.

The recombinant pox viruses of the present invention provide significantadvantages. For example, previously described recombinant vacciniaviruses encoding MUC1 can undergo significant genetic deletion therebydestabilizing the virus, decreasing antigen immunogenicity, and reducingvaccine efficacy. In contrast, the present recombinant pox virusesencode an immunogenic MUC1 fragment that does not undergo significantgenetic deletion, thereby providing an unexpectedly stable andimmunogenic pox virus. Accordingly, efficacy of vaccines including thepresent recombinant pox viruses is substantially increased. Propagationof the present recombinant pox viruses is positively impacted by thestability of the immunogenic MUC1 fragment, e.g., by providing uniformisolation of desired viral strains. Importantly, vaccine efficacyagainst established or pre-existing MUC1 expressing tumors ispotentiated by providing an immunomodulator such as a T-cellco-stimulatory factor, particularly as an admixture with anotherrecombinant pox virus encoding the T-cell co-stimulatory factor.

All publications, patents, and patent applications mentioned in thespecification are indicative of the level of skill of those in the artto which this invention pertains. All publications, patents, and patentapplications are fully incorporated herein by reference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A recombinant pox virus comprising a nucleic acid sequence encodingan immunogenic MUC1 fragment comprising 5 to 25 MUC1 tandem repeatunits, the nucleic acid sequence comprising a first nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:1 that is SEQ ID NO:2; anda second nucleotide sequence encoding 2 to 24 copies of the amino acidsequence of SEQ ID NO:1 wherein, the second nucleotide sequencecomprising 2 to 24 copies of an altered nucleotide-sequence of SEQ IDNO:2 that is altered by changing wobbled nucleotides of the codons ofSEQ ID NO:2.
 2. The recombinant pox virus of claim 1, wherein theimmunogenic MUC1 fragment comprises 6 to 14 tandem repeat units.
 3. Therecombinant pox virus of claim 2, wherein the immunogenic MUC1 fragmentcomprises 9 tandem repeat units.
 4. The recombinant pox virus of claim1, wherein the pox virus is selected from the group consisting oforthopox, suipox and avipox.
 5. A pharmaceutical composition comprisinga recombinant pox virus comprising a nucleic acid sequence encoding animmunogenic MUC1 fragment comprising 5 to 25 MUC1 tandem repeat units,the nucleic acid sequence comprising a first nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:1 that is SEQ ID NO:2; asecond nucleotide sequence encoding 2 to 24 copies of the amino acidsequence of SEQ ID NO:1 wherein, the second nucleotide sequencecomprising 2 to 24 copies of an altered nucleotide sequence of SEQ IDNO:2 is altered by changing wobbled nucleotides of the codons of SEQ IDNO:2; and a third nucleotide sequence encoding an immunomodulator. 6.The pharmaceutical composition of claim 5, wherein the immunomodulatoris selected from the group consisting of T-cell co-stimulatory factorsand cytokines.
 7. The pharmaceutical composition of claim 6, wherein thecytokine is an interleukin.
 8. The pharmaceutical composition of claim5, wherein the immunomodulator is both a T-cell co-stimulatory factorand a cytokine.
 9. The recombinant pox virus of claim 5, wherein the poxvirus is selected from the group consisting of orthopox, suipox andavipox.
 10. The pharmaceutical composition of claim 5, wherein said MUC1fragment comprises about 6 to 14 tandem repeat units.
 11. A method ofgenerating an immune response in a mammal having a MUC1-expressingtumor, the method comprising: (a) administering to the mammal the poxvirus of claim 1 as a first pox virus; and (b) administering an amountof a second pox virus selected from the group consisting of orthopox,suipox and avipox.
 12. The method of claim 11, wherein the second poxvirus is from a viral genus different from said pox virus of step (a).13. The method of claim 11, further comprising administering to themammal an immunomodulator.
 14. A method for generating an immuneresponse in a mammal that contains a MUC1-expressing tumor, the methodcomprising administering to said mammal the pox virus of claim
 4. 15.The recombinant pox virus of claim 1, wherein the pox virus is MVA. 16.The method of claim 13, wherein the immunomodulator is a cytokine or aco-stimulatory molecule.
 17. The method of claim 16, wherein saidco-stimulatory molecule is B7.
 18. The method of claim 17, wherein saidB7 is B7.1 or B7.2.
 19. The method of claim 16, wherein the cytokine isan interleukin.
 20. The method of claim 11, wherein said first pox virusis selected from the group consisting of an orthopox virus vector, anavipox virus vector, a suipox virus vector, and a capripox virus vector.21. The method of claim 20, wherein the first pox virus is an orthopoxvirus.
 22. The method of claim 21, wherein the orthopox virus is avaccinia virus.
 23. The method of claim 21, wherein the vaccinia virusis an MVA.
 24. The method of claim 11, wherein the first pox virus is anorthopox virus and the second pox virus is an avipox virus.
 25. Themethod of claim 24, wherein the avipox virus is a fowipox virus.
 26. Themethod of claim 24, wherein the orthopox virus is a vaccinia virus. 27.The method of claim 26, wherein the vaccinia virus is MVA.
 28. Themethod of claim 11, wherein said first pox virus further comprises anucleic acid sequence encoding an immunomodulator.
 29. The method ofclaim 11 or 28, wherein the second pox virus further comprises a nucleicacid sequence encoding an immunomodulator.
 30. The recombinant pox virusof claim 1, wherein at least one of the copies of an altered nucleotidesequence is selected from the group consisting of SEQ ID NOS: 4–12. 31.The pharmaceutical composition of claim 5, wherein at least one of thecopies of an altered nucleotide sequence is selected from the groupconsisting of SEQ ID NOS: 4–12.
 32. The method of claim 11, wherein atleast one of the copies of an altered nucleotide sequence is selectedfrom the group consisting of SEQ ID NOS: 4–12.
 33. A recombinant poxvirus comprising a nucleic acid sequence encoding an immunogenic MUC1fragment comprising 5 to 25 MUC1 tandem repeat units, the nucleic acidsequence comprising a first nucleotide sequence having SEQ ID NO:2; anda second nucleotide sequence comprising 2 to 24 altered nucleotidesequences encoding 2 to 24 altered tandem repeats, wherein each alteredtandem repeat is altered from SEQ ID NO:2 by changing at least onenucleotide of at least one codon of SEQ ID NO: 2 so that the amino acidof SEQ ID NO: 1 is maintained or by substituting at least one codon inSEQ ID NO:1 such that such substituted codons is selected from the groupconsisting of substituting at least one of the glycines in the SEQ IDNO:1 to serine, substituting at least one of the serines in the SEQ IDNO:1 to glycine, and substituting the valine in the SEQ ID NO:1 toleucine.
 34. A recombinant pox virus comprising a nucleic acid sequenceencoding an immunogenic MUC1 fragment comprising 6 identical amino acidtandem repeat units, the nucleic acid sequence comprising a firstnucleotide sequence encoding the amino acid sequence of SEQ ID NO:1 thatis SEQ ID NO:2; and a second nucleotide sequence encoding 5 copies ofthe amino acid sequence of SEQ ID NO:1 as the other 5 tandem repeatunits, the second amino acid sequence comprising 5 copies of an alterednucleotide sequence of SEQ ID NO:2 by changing wobbled nucleotides ofthe codons of SEQ ID NO:2, the 5 copies encoding the other 5 tandemrepeat units.
 35. A recombinant pox virus comprising nucleic acidsequences encoding 5 to 25 MUC1 tandem repeat units, said tandem repeatunits having an amino acid sequence of SEQ ID NO:1, wherein at least oneof the nucleic acid sequences encoding the tandem repeats has SEQ IDNO:2 and at least one of other nucleic acid sequences encoding thetandem repeats is altered to reduce duplications of codons.
 36. Therecombinant pox virus of claim 35, wherein at least one nucleic acidencoding the tandem repeats is altered by changing wobbled nucleotidesof codons of SEQ ID NO:2.
 37. The recombinant pox virus of claim 35,wherein at least one nucleic acid encoding the tandem repeats is alteredby the third base of threonine codons 3, 11 and 16 of SEQ ID NO: 2 usingACG, ACT, and ACA respectively.